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Cell Disinte NPTEL
Cell Disinte NPTEL
Methods for Cell disruption : The methods of cell disruption used to release the product
depends on the composition of plasma membrane, presence or absence of cell wall,
choice of location and the kind of product (enzymatic/non-enzymatic or simply bioactive
substance). The different methods utilizes a unique mechanism to disturb the integrity of
plasma membrane. These methods can be classify into 3 major section:
1. Physical Methods- These methods play with the physical parameters to damage the
cell to release the product.
B. Osmotic Shock-Most of the mammalian cell have a plasma membrane with active
transporter to maintain the osmotic balance. Maintaining an osmotic balance is an active
process with expenditure of energy. Prolonged exposure to the cell with hypotonic liquid
such as water causes osmotic imbalance and ultimately causes lysis of cell. In this
process due to inflow of water, cell swell and ultimately burst to release the products.
According to the Hoff‟s equation, osmotic pressure Π is directly propotional to the
concentration of solute and temperature.
Π = RT(Ci-Co)………………………………………..Eq 28.1
Each mammalian cell has differential suspectibility towards osmotic shock. Red blood
cells as shown in Figure 28.2, will be lysed with the addition of a tiny drop of water.
Plant or bacterial cells are more resistant towards osmotic lysis due to presence of thick
cell wall.
Figure 28.3: Ultrasonication. (A) Ultrasonic probe (B) Principle of sonic waves mediated cell disruption.
A. Alkali Treatment-This is a harsh but effective chemical treatment to lyse the cells.
Alkali treatment causes lipid saponification which disturbs the lipid packing and affects
the cell wall integrity.
Figure 28.4: Effect of detergent on plasma membrane integrity. (A) Solubilization (B) Permeabilization
D. Enzyme Digestion: Enzymatic methods are specific, gentle and most effective but
costly. Lysozyme is commercially available to treat bacteria to release intracellular
products. In addition to lysozyme, there are three other types of bacteriolytic enzymes,
glycosidases, acetylmuramyl-L-alanineamidase and endopeptidase. Few protease are also
found to be bacteriolytic. Yeast cell lysis requires a mixture of different enzymes such as
glucanase, protease, mannanase or chitinase. Plant cells can be lysed by cellulose and
pectinase. In most of the enzyme mediated cell lysis method, the rupture of cell wall
depends on the osmotic pressure of the external medium. In few cases, enzymatic
digestion is performed to remove external cell wall and then in second step protoplast is
disrupted by gentle agitation.
𝑪𝐦𝐚𝐱
𝐥𝐧( ) = −𝐤𝐭
𝑪𝐦𝐚𝐱 − 𝑪
Here, Cmax is the concentration of product that can be released from a given amount of
cell suspension, C is the concentration of product released at a given time “t” and k is the
first order constant. This relationship holds true only for batch mode of operation. The
value of k depends on type of impeller, bead size and loading, speed of agitation and
temperature.
𝟏 𝟐
𝐏𝐬 = 𝐩𝐯
𝟐
Ps is dynamic pressure, v is jet velocity and p is the density of fluid.
Cell disruption in high pressure homogenizer and release of a produce is a first order
kinetics and it may be given by first order equation
𝑪𝒎𝒂𝒙
) = 𝒌𝑵
𝑪𝒎𝒂𝒙 − 𝑪
Here, N is number of passes through the valve and and k is the first order constant. As
high pressure homogenization passes the cell at a very high speed through a narrow
valve, it disrupt the cells and simulatenously it lower down the temperature as well.
Physical Properties
1. Molecular weight
2. Boiling point (in case both are liquid, as in this case)
3. Freezing point
4. Crystallization
5. Solubility
6. Density
Chemical Properties
1. Functional Group, for example, phenol has –OH where as aniline has NH2.
Now for example you have a mixture of compound 1 (benzene) and compound 3
(Aniline) and you would like to purify benzene rather than aniline. In this situation, you
can take the physical and chemical properties of benzene into the account and isolate it
from the mixture.
Figure 29.1: Chemical Structure and physical Properties of benzene, phenol and aniline.
Concentration in Phase A
𝐾𝑑 =
Concentration in Phase B
Similarly one can also exploit other physical & chemical parameters as well. With each
and every physical and chemical parameter the molecule present in the mixture will
distribute as per their behavior in each parameter.
Chromatogram: The plot of elution volume along with the absorbance is known as
chromatogram as given in Figure 29.4. The volume or time it takes for a analyte to come
out from the column is known as retention volume or time. The chromatogram may have
separate peaks (A and B) or peaks (C and D) with overlapping base, these peaks are
called fused peaks.
Resolution: The ability of a chromatography column to separate two analyte peak from
another is known as resolution. It is defined as the ratio of difference in retention time
between two peaks and average of base of peak width. It is given by
⍙tR
𝑅𝑠 =
Wav
When Rs=1, the separation of two peaks is 97.7% and a column with Rs more than 1.5
considered good. The number of distribution events govern the ability of a column to
separate the two analytes. In another words, resolution is directly proportional to the
number of distribution events. In column chromatography, each thin plain of column
matrix participate in distribution of molecule. Assume height of a distribution plain is H
and length of a column is L, hence number (N) of distribution plain in a column is given
by,
L
𝑁=
H
N=16 (tR/W)2
N=5.54 (tR/W1/2)2
(1) As number of distribution plain will go up, it will allow the analyte to travel for
longer period of time, consequently it will increase the distance between two peaks.
(2) As number of distribution plain will go up, it will reduce the width of the base of
peak, as a result the peaks will be more sharp. A representative example, how number of
distribution plain affects the base of the peak is given in Figure 29.5. As the number is
increasing, the peak width is decreasing. Hence, number of distribution is an indirect way
to measure the column efficiency, higher N number is desirable for better separation.
Figure 29.5: Relationship between number of distribution planes (N) and peak width.
2. Pump: One or two pump to flow the buffer from reservoir. Different types of pumps
are used in chromatography system, mostly based on the pressure level required to
perform chromatography.
3. Mixer: A mixer is required to mix the buffer received from both pumps to form a linear
or step gradient.
5. Detector: The elution coming out from column goes to the online monitoring system to
test the presence of the analyte based on different properties. There are different types of
detectors are known in chromatography such as UV-Visible detector etc.
7. Recorder: The profile of eluent with respect to the measured property in a detector can
be plotted in the recorder.
Principle: This chromatography distributes the analyte molecule as per charge and their
affinity towards the oppositively charged matrix. The analytes bound to the matrix are
exchanged with a competitive counter ion to elute. The interaction between matrix and
analyte is determined by net charge, ionic strength and pH of the buffer. For example,
when a mixture of positively charged analyte (M, M+, M-1, M-2) loaded onto a positively
charged matrix, the neutral or positively charged analyte will not bind to the matrix
where as negatively charged analyte will bind as per their relative charge and needed
higher concentration of counter ion to elute from matrix (Figure 30.1).
Figure 30.1: Affinity of analytes (M, M+, M-1, M-2) towards positively charged matrix.
The matrix used in ion-exchange chromatography is present in the ionized form with
reversibly bound ion to the matrix. The ion present on matrix participitate in the
reversible exchange process with analyte. Hence, there are two types of ion-exchange
chromatography:
Isoelectric point and charge on a protein: Protein is a polymer made up of amino acids
with ionizable side chain. At a particular pH, these amino acid side chain ionizes
differentially to give a net charge (positive/negative) to the protein. The pH at which the
net charge on a protein is zero is called as Isoelectric point (pI). The protein will have a
net positive charge below the pI where as it has net negative charge above the pI value
(Figure 30.3).
1. pI value and Net charge- The information of a pI will be allow you to calculate the
net charge at a particular pH on a protein. As discussed above, a cation exchange
chromatography can be use below the pI where as an anion exchange chromatography
can be use above the pI value.
2. Mobile Phase-The ionic strength and pH are the crucial parameters to influence the
property of the mobile phase.
3. Sample Preparation- The sample is prepared in the mobile phase and it should be free
of suspended particle to avoid clogging of the column. The most recommended method to
apply the sample is to inject the sample with a syringe.
4. Elution- There are many ways to elute a analyte from the ion-exchange column. (1)
Isocratic elution (2) Step-wise gradient (3) Continuous gradient either by salt or pH (4)
affinity elution (5) displacement chromatography
Figure 30.4 : Operation of the Ion-exchange Chromatography. (A) Chromatography system to perform gradient elution of
analytes to give an (B) elution profile.
Figure 31.1: Folding of Protein in an aqueous environment. Following a series of folding stages, protein adopts a 3-D
conformation with hydrophobic patches present in the core.
Addition of low amount of salt to the protein solution results in the displacement of
bonded water molecule with an increase in protein solubility (Figure 31.2). This effect is
called as “salting-in”. In the presence of more amount of salt, water molecule shielding
protein side chains are displaced completely with an exposure of hydrophobic patches on
protein surface to induce protein precipitation or decrease in protein solubility. This
effect is called as “salting-out”. The phenomenon of salting out is modulated so that
addition of salt induces exposure of hydrophobic patches on protein but does not cause
precipitation or aggregation. The exposure of hydrophobic patches facilitates the binding
of protein to the non-polar ligand attached to the matrix. When the concentration of salt is
decreased, the exposed hydrophobic patches on protein reduces the affinity towards
matrix and as a result it get eluted (Figure 31.3).
Figure 31.2: Effect of salt on protein, salting in and salting out effect.
The choice of HIC gel-The different commercially available HIC matrix are given in
Table 31.1. Choosing a suitable HIC matrix is essential to achieve best result. The
strength of the binding of analyte on a HIC column is governed by the length of the
aliphatic linear ligand. Matrix with aromatic ring containing ligand makes additional Π-Π
interaction and they will bind analyte more strongly than same number of carbon
aliphatic ligand. In addition, presence of Π-Π interaction gives selectivity as well, such as
ring containing aromatic ligand, phenylalanine. At last, ligand density plays a vital role in
the strength of binding of an analyte to the matrix. Hence, these points should be consider
to choose a suitable matrix for purification.
1 Butyl-S-Sepharose -Butyl
2. Sample Preparation- The sample is prepared in the mobile phase and it should be free
of suspended particle to avoid clogging of the column. The most recommended method to
apply the sample is to inject the sample with a syringe.
3. Elution- There are many ways to elute a analyte from the hydrophobic interaction
column. (1) decreasing salt concentration, (2) changing the polarity of the mobile phase
such as alchol, (3) By a detergent to displace the bound protein.
Figure 32.1: Gel Filtration Matrix has Beads with different pore sizes.
Suppose the total column volume of a gel is Vt and then it is given by-
Vt=Vg+Vi+Vo………………………………………………Eq (32.1)
Vg is the volume of gel matrix, Vi is the pore volume and Vo is the void volume. The
volume of mobile phase flow to elute a column from a column is known as elution
volume (Ve). The elution volum is related to the void volume and the distribution
coefficient Kd as given below
Ve=Vo+KdVi……………………………………………….Eq (32.2)
𝑽𝒆−𝑽𝒐
𝐊𝐝 = 𝑽𝒊
……………..……………………………….Eq (32.3)
Kd is the ratio of inner volume available for an analyte and it is independent to the
column geometry or length. As per relationship given in Eq 32.3, three different type of
analytes are possible:
1. Analyte with Kd=0, or Ve=Vo, these analytes will be completely excluded from the
column.
2. Analyte with Kd=1 or Ve=Vo+Vi, these analytes will be completely in the pore of the
column.
3. Analyte with Kd>1, in this situation analyte will adsorb to the column matrix.
Choice of matrix for gel filtration chromatography-The choice of the column depends
on the range of molecular weight and the pressure limit of the operating equipment. A list
of popular gel filtration column matrix with the fractionation range are given in Table
32.1.
2. Sample Preparation- The sample is prepared in the mobile phase and it should be free
of suspended particle to avoid clogging of the column. The most recommended method to
apply the sample is to inject the sample with a syringe.
3. Elution- In gel filtration column, no gradient of salt is used to elute the sample from
the column. The flow of mobile phase is used to elute the molecules from the column.
4. Column Regeneration- After the analysis of analyte, gel filtration column is washed
with the salt containing mobile phase to remove all non-specifically adsorb protein to the
matrix. The column is then equiliberated with mobile phase to regenerate the column.
The column can be store at 40C in the presence of 20% alchol containing 0.05% sodium
azide.
6 Sepharose 4B 60,000-20,000,000
7 Sepharose 6B 10,000-4,000,000
The molecular weight and size of a protein is related to the shape of the molecule and the
relationship between molecular weight (M) and radius of gyration (Rg)is as follows-
Rg ∝ Ma……………………………………………………………………Eq 32.4
here “a” is a constant and it depends on shape of the molecule, a=1 for Rod, a=0.5 for
coils and a=0.33 for spherical molecules.
The set of known molecular weight standard protein can be run on a gel filitration
column and elution volume can be calculated from the chromatogram (Figure 32.4). A
separate run with the analyte will give elution volume for unknown sample. Using
following formula, Kd value for all standard protein and the test analyte can be
𝑽𝒆−𝑽𝒐
calculated. 𝐊𝐝 = 𝑽𝒊
A plot of Kd versus log mol wt is given in Figure 32.4, B and it will allow us to calculate
the molecular weight of the unknown analyte.
Figure 32.3: Determination of molecular weight by gel filtration chromatography. (A) Gel filtration chromatogram with the
standard proteins (1-6), (B) Relationship between distribution constant (Kd) and Log Molecular weight.
Besides determining molecular weight of the protein as discussed above, gel filutration
chromatography can be used to determine following properties of a protein:
1. Oligomeric status
2. Desalting
3. Protein-ligand interaction
4. Folding pathway.
R + L ⇋ RL
𝐑 [𝐋]
𝐊𝐝 = …………………….33.1
[𝐑𝐋]
Choice of matrix for Affinity chromatography- Different popular affinity matrix used
for protein purification is given in Table 33.1. The choice of matrix solely depends on the
affinity tag present on the recombinant protein produced after genetic engineering.
2. Sample Preparation- The sample is prepared in the mobile phase and it should be free
of suspended particle to avoid clogging of the column. The most recommended method to
apply the sample is to inject the sample with a syringe.
3. Elution- There are many ways to elute a analyte from the affinity column. (1)
increasing concentration of counter ligand, (2) changing the pH polarity of the mobile
phase, (3) By a detergent or chaotrophic salt to partially denature the receptor to reduce
the affinity for bound ligand.
4. Clinical diagnosis
5. Estimation of biomolecules
6. Immunoassay
If the distance travelled by a molecule on TLC plate is Dm where as the distance travelled
by the solvent is Ds, then the retardation factor (Rf) of molecule is given by:
Preparation of TLC plate- A silica slurry is prepared in water and spread on the glass or
alumina sheet as a thin layer and allowed to dry. It is baked at 1100C for 1hr in a hot air
oven and then the plate is ready for TLC. The layer is thin (~ 0.1-0.25 mm) for analytical
application and thick (0.4-2.1 mm) for preparative or bio-assay purposes.
Spoting: The events involved in spotting is given in Figure 34.3. A line is draw with a
pencil little away from the bottom. Sample is taken into the capillary tube or in a pipette.
Capillary is touched onto the silica plate and sample is allowed to dispense. It is
important that depending on the thickness of the layer, a suitable volume should be taken
to apply. Spot is allowed to dry in air or a hair dryer can be used instead.
Running of the TLC: Once the spot is dried, it is placed in the TLC chamber in such a
way that spot should not be below the solvent level. Solvent front is allowed to move
until the end of the plate.
Analysis of the chromatography plate- The plate is taken out from the chamber and air
dried. If the compound is colored, it forms spot and for these substances there is no
additional staining required. There are two methods of developing a chromatogram-
Staining procedure- In the staining procedure, TLC plate is sprayed with the staining
reagent to stain the functional group present in the compound. Forx. Ninhydrin is used to
stain amino acids.
1. Autoradiography- A TLC plate can be placed along with the X-ray film for 48-72 hrs
(exposure time depends on type and concentration of radioactivity) and then X-ray film is
processed.
1. Tailing effect-In general sample forms round circular spot on the TLC plate. It is due
to the uniform movement of the solvent front through out the plate. But in few cases
instead of forming a spot, a compound forms a spot with long trail or rocket shape spot
(Figure 34.5). it is due to few reasons as given below:
A. Over-loading- if the sample is loaded much more than the loading capacity of the
TLC plate, it appears as spot with trail or rocket shape spot. A diluted sample can be
tested to avoid this.
2. No movement of sample- In few cases, a sample doesn‟t move from the spot after the
run is completed. These problems are common with high molecular weight substances
such as protein or chemicals with large number of functional group. In this case, a change
in polarity or pH of solvent system can be explored to bring the compound into the
solvent front so that it run on silica plate to get resolved.
3. Movement is too fast-In few cases, the movement of a compound is too fast and does
not give time to interact with the matrix to resolve into individual compounds. In this
case, a change in polarity of solvent system can be explored to retard the running of the
sample.
5. Estimation of biomolecules
6. Bio-assay
Suppose a charged particle has net charge Q and the external electric field is E, then the
force F responsible for giving electrophoretic mobility,
F=
Q.E…………………………………………….Eq (35.1)
The friction forces F which is opposing the movement of the charged particle is as
follows
F= ƒ.
v……………………………………………Eq (35.2), here ƒ is the friction coefficient
and the v is the velocity of the electrophoretic mobility. The movement of a spherical
through a liquid medium (gel) of the viscosity η, the friction coefficient ƒ is given by :
ƒ = 6Πηrv……………………………………………Eq (35.3)
𝐐
The electrophoretic mobility v is given by: 𝒗 = 𝟔𝚷𝛈𝐫
As Q=ze, where z is the valency and e is the electronic charge, the electrophoretic
mobility can be expressed as:
𝐳𝐞
𝒗=
𝟔𝚷𝛈𝐫
Hence, electrophoretic mobility v is directly proportional to the charge and inversely
proportional to the viscosity of the medium, size and shape of the molecule. In the case of
relative mobility, it is directly related to the charge/radius of the molecule. For a globular
protein, the radius (r) of the molecule is related to the molecular mass of the
macromolecule. The relative mobility, v’ is as follows
𝐂𝐡𝐚𝐫𝐠𝐞
𝒗′ = ………………………………………Eq (35.4)
𝐦𝐚𝐬𝐬
Gel electrophoresis:
Buffer and reagent for electrophoresis- The different buffer and reagents with their
purpose for vertical gel electrophoresis is as follows-
1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide
polymerization.
7. Sodium dodecyl sulphate-it is used to denature and provde negative charge to the
protein.
Casting of the gel: The acrylamide solution (a mixture of monomeric acrylamide and a
bifunctional crosslinker bisacrylamide ) is mixed with the TEMED and APS and poured
in between the glass plate fitted into the gel caster. Ammoinum persupfate in the presence
of TEMED forms oxygen free radicals and induces the polymerization of acryalide
monomer to form a linear polymer (Figure 35.3). These linear monomers are
interconnected by the cross linking with bis-acrylamide monomer to form a 3-D mesh
with pores. The size of pore is controlled by the concentration of acrylamide and amount
of bis-acrylamide in the gel. IN a vertical gel electrophoresis system, we cast two types of
gels, stacking gel and resolving gel. First the resolving gel solution is prepared and
poured into the gel cassette for polymerization. A thin layer of organisc solvent (such as
butanol or isoproponal) is layered to stop the entry of oxygen (oxygen neutralizes the free
radical and slow down the polymerization) and make the top layer smooth. After
polymerization of the resolving gel, a stacking gel is poured and comb is fitted into the
gel for construction of different lanes for the samples (Figure 35.4).
Figure 35.4: Different steps in performace of vertical gel electrophoresis to resolve sample.
Running of the gel: The sample is prepared in the loading dye containing SDS, β-
mercaptoethanol in glycerol to denature the sample and presence of glycerol facilitates
the loading of sample in the well. As the samples are filled vertically there is a distance
drift between the molecules at the top Vs at the bottom in a lane. This problem is taken
care once the sample run through the stacking gel. The pH of the stacking gel is 6.8 and
at this pH, glycine is moving slowly in the front where as Tris-HCl is moving fast. As a
result, the sample gets sandwiched between glycine-Tris and get stacked in the form of
thin band. As the sample enters into the resolving gel with a pH 8.8, the glycine is now
charged, it moves fast and now sample runs as per their molecular weight (due to SDS
they have equal negative charge). After tracking dye reaches to the bottom of the gel, gel
is taken out from the glass plate with the help of a spatula and it is stained with coomassie
brilliant blue R250 dye. The dye stains protein present on the gel. A typical SDS-PAGE
is given in the Figure 35.5.
In SDS-PAGE, the relative mobility and the log molecular weight as given by
𝐀−𝐥𝐨𝐠 𝐌
𝒗′ = 𝐕𝐨 ………………………………………Eq (35.5)
𝐀
Molecular weight of a protein can be determined by plotting relative migration Rf
with the log molecular weight of standard protein.
Figure 35.5: Determination of molecular weight using SDS-PAGE. (A) SDS-PAGE (B) Determinaion of Rf.
Summary of previous lecture: In the previous lecture, we discussed the principle of the
electrophoresis. It is a mechanism of separating the charged species. In addition, we
discussed the different type of electrophoresis techniques, details of vertical gel
electrophoresis, instrument, reagent and performing the SDS-PAGE.
2. Native PAGE: SDS-PAGE discussed in the previous lecture is using anionic detergent
sodium dodecyl sulfate and β-mercaptoethanol to give equal charge to all protein and
breaks the disulphide linkage. As a result, the 3-D structure of the protein is destroyed
and it migrate as per their subunit molecular weight. In the native PAGE, sample is
prepared in the loading dye does not contains detergent or denaturating agent and as a
result, sample runs on the basis of charge/mass. In native PAGE, the 3-D conformation as
well as activity of the protein remains unaffected.
3. Urea PAGE: In this method, insoluable protein is dissolved in Urea and samples
separate based on their charge/subunit mass. A gradient Ura PAGE is used to monitor the
folding states of a protein.
Buffer and reagent for electrophoresis- The purpose of each reagents used in
horizontal gel electrophoresis are as follows-
1. Agarose-polymeric sugar used to prepare horizontal gel for DNA analysis.
2. Ethidium bromide- for staining of the agarose gel to visualize the DNA.
Casting of the agarose gel- Different steps to cast the agarose gel for horizontal gel
electrophoresis are given in Figure 36.2. The agarose powder is dissolved in a buffer
(TAE or TBE) and heated to melt the agarose. Hot agarose is poured into the gel cassette
and allowed it to set. A comb can be inserted into the hot agarose to cast the well for
loading the sample. In few cases, we can add ethidium bromide within the gel so that it
stains the DNA while electrophoresis.
Figure 36.2: Different steps in casting of the agarose gel for horizontal gel electrophoresis apparatus.
Running and staining-The gel cassette is placed in the electrophoresis tank submerged
completely and DNA loaded into the well with the help of pipetman and run with a
constant voltage. DNA runs from negative to positive end and ethidium bromide (EtBr)
present in the gel stain the DNA. Observing the agarose gel in a UV-chamber shows the
DNA stained with EtBr as organe colored fluorescence (Figure 36.3).
Plasmid DNA
RNA
To study the DNA-protein interaction, a fix amount of DNA is incubated with the
increasing concentration of protein (Figure 36.4). Due to the formation of DNA-protein
complex, the hydrodynamic volume of the complex increases and a shift in band is
observed. The DNA has a extended structure and it provides docking site for several
protein molecules such as single stranded binding protein (SSB). As a result, a gradual
shift in DNA band will be observed until the DNA binding site is not saturated with the
protein molecules. Hence, at the end of the experiment, we can be able to understand
several aspects of DNA-protein interaction:
1. Whether protein-X has a affinity for DNA and the interaction is specific or non-
specific in nature.
2. What will be affinity parameters of the interaction of DNA to protein in making DNA-
protein complex?
4. Southern blotting- In southern blotting, the genomic DNA is digested with the EcoRI
or BamHI and the DNA fragments are resolved on the agarose gel. The gel is incubated
in an alkaline solution to denature the double stranded DNA to single stranded form.
DNA is transferred on the nitrocellulose membrane by capillary action by applying a
uniform pressure either by suction pressure or by placing wet paper towels. The
membrane is incubated with a non-specific DNA such as sonicated calf thymus genomic
DNA to block the binding sites on the membrane. A single stranded radioactive probe is
added to the membrane and allowed to bind. Membrane is washed and the blot is
developed by autoradiography. The DNA fragment complementary to the probe sequence
binds the radioactive probe and give positive signal (Figure 36.6).
DNA sequencing-Historically there are two methods of DNA sequencing with a similar
principle of breaking the DNA (chemical or enzymatic method) into the small fragment
followed by separation and analyze them on a high resolution electrophoresis gel.
Protocol for Di-deoxy sequencing- There are two protocols people adopt to sequence
DNA following di-deoxy chain termination method (Figure 37.2).
Original sanger protocol uses klenow fragment as polymerase for DNA synthesis where
as termination protocol uses a T7 polymerase or sequenase. The DNA sequencing by
original sanger protocol has following steps:
Step 4: DNA synthesis continues until terminated by the incorporation of the specific
ddNTPs (either A, T, G or C).
Step 2: A limited amount of NTPs are added along with the one of the radiolabeled
nucleotide to label the DNA through the length.
Step 4: The polymerase reaction continues with 4 nucleotide and one ddNTPs. Synthesis
is terminated at the specific ddNTPs (either A, G. C. T) to give DNA fragment of
different length.
The polymerization reaction is analyzed on a high resolution polyacrylamide gel. The use
of sequenase allow to perform sequencing of long DNA stretches where as original
sanger method is more appropriate for short length DNA.
Maxam-Gilbert method: DNA cloning and polymerization reactions made the sanger
method less popular than maxam-gilbert DNA sequencing method. This method was
discovered by Allan Maxam and Walter Gilbert in 1977 which is based on chemical
modification and subsequent cleavage. In this method, a 3‟ or 5‟ radiolabeled DNA is
treated with a base specific chemicals which randomly cleaves the DNA at their specific
target nucleotide. These fragments are analyzed on a high resolution polyacrylamide gel
and a autoradiogram is developed (Figure 37.3). The fragment with terminal radiolabel
appears as band in the gel. The chemical reactions are performed in two steps;
Base Specific Reaction: Different base specific reagents are used to modify the target
nucleotide.
Reaction 1: Dimethylsulfate (DMS) modifies the N7 of guanine and then opens the ring
between C8 and N9.
Reaction 4: Where as in the presence of salt (NaCl), it breaks the ring of cytosine.
Cleavage reaction : After the base specific reactions, piperidine is added which will
replace the modified base and catalyzes the cleavage of phsophodiester bond next to the
modified nucleotide.
The sequencing of a long protein has multiple stages: A protein needs to go through
following stages for elucidation of its sequence as well as bonding pattern. These stages
are schematically given in Figure 38.1. Over-all, the complex protein first needs to break
into the subunits, and sequential release of amino acids from N-terminus of each
fragment following edman-degradation method. At the end, the sequence of each
fragment can be put together to deduce the complete amino acid sequence of protein. The
details of each stage is as follows-
Stage 1. Breaking Disulphide Bonds: In protein two cysteine amino acids are linked by
a disulphide linkage. The disulphide linkage interfere with the complete sequencing
procedure as it doesn‟t allow the release of cleaved amino acid from the peptide chain.
There are two approaches two disrupt the disulphide linkage in a protein sequence
(Figure 38.2). In first approach, protein is oxidized with a performic acid to produce two
cysteic acid residues. In another approach, protein is reduced by dithiothreitol (DTT) or
β-mercaptoethanol (β-me) to form two cysteine followed by treatment with iodoacetate to
form carboxymethyl-cysteine. Formation of carboxymethyl-cysteine stops the re-
formation of disulphide bond.
Stage 2. Cleavage of the polypeptide chain: Proteases and the chemical agents targeting
proteins have a specific recognition sequence and they cleave after a particular amino
acid. A list of protease and chemicals commonly used to digest the polypeptides into the
small peptide fragment is given in Table 38.1.
A. Identifying the N-terminal residue: The N-terminal amino acid analysis is a 3 steps
process.
1. Similar to sanger reagent, phenylisothiocynate reacts with the terminal amino group to
form a cyclic phenylthiocarbamoyl derivative.
2. Under acidic condition, the terminal amino acid is cleaved from the main chain as
thiazolinone derivative.
4. PTH-amino acid acid complex can be identify by HPLC or TLC and comparison with
the standard amino acids.
5. Step 1-4 can be repeated again with the next amino acid residue in the peptide chain.
C. C-terminal residues: Not many methods are developed for c-terminal amino acid
analysis. The most common method is to treat the protein with a carboxypeptidase to
release the c-terminal amino acid and test the solution in a time dependent manner.
Stage 4. Ordering the peptide fragments: The usage of different protein cleavage
reagent produces over-lapping amino acid stretches and these stretches can be used to put
the whole sequence.
Stage 5. Locating disulfide bonds: The protein cleavage by typsin is performed with or
without breaking di-sulphide linkage. Amino acid sequence analysis of the fragments will
provide the site of disulphide bond. The presence of one disulphide will reduce two
peptide fragment and will appear as one large peptide fragment.